This invention relates to methods and apparatus for determining concentrations of active populations of enzymes or enzyme-containing populations, such as bacteria, yeasts, or the like.
Traditionally, such populations were counted by microscopic techniques or by more sophisticated techniques, such as the measurement of optical turbidity of liquids containing the populations. In U.S. Pat. No. 3,506,544 there is described a technique for measuring the concentration of certain constituents of enzyme-catalyzed reactions using a electrochemically-reversible redox couple. The enzyme, a redox couple such as methylene blue, a substrate such as glucose, and a compatible conductive medium such as a buffered water solution, were mixed in an electrolytic cell through which a current was passed by impressing a voltage across a pair of electrodes. The electrodes were typically made of noble metal. The activity of the enzyme reduced the oxidized redox couple at a rate corresponding to the concentration of the enzyme. The reduced form of the redox couple was subject to reoxidization at the anode of the cell, which thus produced a cell current proportional to the concentration of the reduced form ofthe redox couple. The rate of increase in current was thus shown to be proportional to the concentration of enzyme in the solution. In commonly-assigned application Ser. No. 628,514, filed July 6, 1984, a similar technique is described in which a high density, partially-insulated graphite electrode is used. The noble metal or graphite electrodes are rotated rapidly to provide a thin diffusion layer between the electrode surface and the main body of solution.
The various prior art techniques for measuring enzymes or bacteria populations suffered certain deficiencies. Some were not sufficiently sensitive to detect very low concentrations, or were very slow, or required too many steps to make the determinations efficiently. Some were also quite expensive, especially those which used noble metal or similar electrodes.
Also, the prior art apparatus and methods are generally directed to systems which are relatively complex and difficult to operate, and they use relatively bulky, expensive equipment. It has long been felt desirable to provide the art with compact disposable devices which could be used and discarded, in whole or in part.